Method for producing a biosensor

Abstract: A biosensor for rapid quantification of a specific component contained in various biological samples with high accuracy has an electrically insulating base, an electrode system including a working electrode and a counter electrode formed on one face of the insulating base, and a reaction layer formed on the insulating base in close contact with the electrode system. The reaction layer contains at least a hydrophilic polymer, a buffer and an enzyme which is separated from the buffer.

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a biosensor, capable of rapidly quantifying a specific component in a sample solution with high accuracy in a simplified manner. 2. Description of the Related Art Various types of biosensors have heretofore been proposed as a system for quantifying the specific component in the sample solution without requiring diluting or stirring of the sample solution. As an example of such biosensors, a glucose sensor will be described in the following paragraphs. In general, a system combining glucose oxidase with an enzyme electrode or a hydrogen peroxide electrode is already known as a method of quantifying glucose utilizing the enzyme electrode. The glucose oxidase selectively oxidizes a substrate, i. e., β-D-glucose into D-glucono-δ-lactone by using oxygen as an electron acceptor. During this reaction, oxygen is reduced into hydrogen peroxide. By measuring the amount of the oxygen consumed in this reaction by an oxygen electrode, or by measuring the amount of the hydrogen peroxide produced in this reaction by a hydrogen peroxide electrode which utilizes a platinum electrode or the like, the glucose in the sample solution can be quantified. By the above-mentioned method, the measurement is however adversely influenced with a concentration of the dissolved oxygen depending on the subject of the measurement. Further, the measurement is made completely impossible under a condition lacking oxygen. A type of the glucose sensor that does not use oxygen as the electron acceptor but uses a metal complex or an organic compound such as potassium ferricyanide, a derivative of ferrocene or a derivative of quinone as the electron acceptor has therefore been developed. With this type of biosensor, by oxidizing a reductant of the electron acceptor produced as the result of the enzyme reaction by the electrode, the concentration of the glucose can be determined based on the current consumed for this oxidation reaction. This manner of measurement is not limited to glucose but has been widely applied to the quantification of any other substrate than glucose. As an example of this type of biosensor, a glucose sensor is known (Japanese Laid-Open Patent Publication No. Hei 1-114,747) which will be described below. The disclosed biosensor has a configuration comprising an electrical insulating base provided with an electrode system including a working electrode and a counter electrode, a filter layer composed of polycarbonate porous film, an electron acceptor carrying layer, an enzyme carrying layer, a buffer carrying layer, and a developing layer composed of woven cellulose, which are sequentially laminated on the insulating base by placing some space from the electrode system. In this configuration, the above-mentioned carrying layers are prepared by impregnating cellulosic porous films with aqueous solutions of the electron acceptor, the enzyme, and the buffer, and then drying the impregnated bodies. The operation of this glucose sensor is as follows. The sample solution titrated on the developing layer is first passed to the buffer carrying layer, whereby pH value of the sample solution is adjusted to a pH value that can give the highest activity to the enzyme by the buffering action of the buffer. Next, the glucose in the sample solution reacts specifically with the glucose oxidase in the enzyme carrying layer. At the same time, the electron acceptor, such as potassium ferricyanide in the electron acceptor carrying layer, is reduced by the electron produced by the above-mentioned reaction to produce potassium ferrocyanide. The amount of the produced potassium ferrocyanide is directly proportional to the concentration of glucose contained in the sample solution. After the substances having a large molecular weight such as protein which disturb the electrode reaction contained in the sample solution are filtered off by the filter layer, the sample solution reaches the electrode system provided on the insulating base. In order to prevent erroneous measurement, part of the electrode system is covered with the insulating layer. By measuring the value of the current for oxidizing the potassium ferrocyanide produced in the sample solution by the electrode system, it is possible to determine the glucose concentration of the sample solution. In the configuration of such prior art sensors, however, there is an inconvenience that an adverse influence is given to the responsive current, because wetting of the surface of the insulating base including the electrode system with the sample solution is not necessarily uniform and thus bubbles are retained between the porous body of the filter layer and the insulating base. Further, if the sample solution contains substances liable to be absorbed in the electrode or substances having an electrode activity, there would be a case wherein the response of the sensor is adversely influenced. As a method for overcoming the above-mentioned inconveniences, the following biosensor is proposed and disclosed in Japanese Laid-Open Patent Publication No. Hei 2-062,952. In the disclosed configuration, the sensor comprises an electrically insulating base, an electrode system composed of a working electrode, a counter electrode and a reference electrode formed on the insulating base by means of screen printing or the like, and a reaction layer including a hydrophilic polymer, an oxido-reductase, an electron acceptor, and a buffer as well if required, formed on the electrode system in a manner that the reaction layer is in close contact with the electrode system. When the sample solution containing the substrate is titrated on the reaction layer, the reaction layer dissolves in the sample solution which is thereby adjusted to a pH value at which the highest enzyme activity is achieved by the buffering action of the buffer, the enzyme reacts with the substrate, and the electron acceptor is reduced. After the completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized, and the concentration of the substrate contained in the sample solution is derived from the value of the current consumed for oxidizing the electron acceptor. In the above-mentioned configuration of the prior art sensor, if the biosensor is moistened, the buffer would be partly mixed with the enzyme to induce a buffer would be partly mixed with the enzyme to induce a chemical interaction, thereby lowering the enzyme activity and deteriorating the storing property of the biosensor. In the document EP-A-0 502 504 has been disclosed a biosensor utilizing an enzyme and also a method for producing the same. The biosensor described in this reference determines an analyte (e. g. fructose) contained in a sample liquid by reducing electron acceptors using electrons generated in a reaction of the analyte with an enzyme and then by measuring the reduced amount of the electron acceptors electrochemically. SUMMARY OF THE INVENTION Object of the present invention is to provide a method for producing a biosensor that can be stored for a long period of time and can be utilized in quality control of foodstuffs as well as in clinical tests. The present invention provides a method for producing a biosensor which comprises the steps of: forming a first layer containing an enzyme and a hydrophilic polymer by using water as the medium on a surface of an insulating base in close contact with an electrode system including a working electrode and a counter electrode which are provided on the said insulating base; and forming a second layer comprising a buffer and an amphipathic lipid, wherein said step of forming the second layer comprises spreading an organic solvent solution of said amphipathic lipid on the first layer by using an organic solvent that does not dissolve the hydrophilic polymer contained in the first layer, and drying the spread solution, said organic solvent solution being dispersed with said buffer. In a preferred embodiment of the above-mentioned method, the step of forming the first layer comprises spreading an aqueous solution which dissolves the enzyme and the hydrophilic polymer on the insulating base and drying the spread solution. It is preferable that the above-mentioned aqueous solution which dissolves the enzyme and the hydrophilic polymer further dissolves an electron acceptor. While novel features of the invention are set fourth in the preceding, the invention, both as to organization and content, can be further understood and appreciated, along with other objects ad features thereof, from the following detailed description and example when taken in conjunction with the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a cross-sectional side view showing an essential part of a biosensor prepared in accordance with Example 1 of the present invention. Fig. 2 is an exploded perspective view of the biosensor shown in Fig. 1 removed of its reaction layer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following paragraphs, embodiments of the method for producing a biosensor in accordance with the present invention will be described in detail with reference to the attached drawings. As described in the above, the biosensor produced according to the method of the present invention has a configuration that the reaction layer formed on the electrode system on the insulating base is in close contact with the electrode system, and contains the hydrophilic polymer and the enzyme, which latter is separated from the buffer. Further, by separating the enzyme from the buffer it is possible to prevent a partial mixing of the buffer with the enzyme attributable to a possible wetting or moistening of the biosensor and a lowering of the activity of the enzyme attributable to the chemical interaction induced by the mixing, and thus to maintain the enzyme at a condition that stabilizes the enzyme during the storing period of the biosensor. In the biosensor prepared in accordance with the method of the present invention, the layer containing the buffer is in close contact with the layer containing the enzyme. The biosensor having the above-mentioned configuration can be obtained by the following manufacturing process. The process of the present invention comprises steps of forming a first layer composed of the enzyme and the hydrophilic polymer on the insulating base, which is in close contact with the electrode system, by using water as a medium, and forming a second layer containing the buffer and an amphipathic lipid on the first layer by using an organic solvent as the medium that does not dissolve the hydrophilic polymer contained in the first layer. The biosensor produced according to the method of the present invention has a layer containing an amphipathic lipid that facilitates an infusion of the sample solution into the reaction layer. In addition to lecithin (phosphatidyl cholin) used in the following examples, further amphipathic (lipophilic and hydrophilic) lipids such as phospholipids, exemplified as phosphatidyl serin, phosphatidyl ethanolamine and the like, are preferable as the amphipathic lipid. As the hydrophilic polymer for forming the reaction layer, in addition to carboxymethyl cellulose and polyvinyl pyrrolidone which are used in the following examples, there are exemplified polyvinyl alcohol, water soluble cellulose derivatives such as ethyl cellulose and hydroxypropyl cellulose; gelatin, polyacrylic acid and its salts, starch and its derivatives, maleic anhydride and its salts, polyacrylamide, methacrylate resin, poly-2-hydroxyethyl methacrylate. Although the description on the following examples is limited to the two-electrode system composed only of the working electrode and the counter electrode, a more accurate measurement can be performed by employing a three-electrode system also including a reference electrode. In addition to potassium ferricyanide used in the following examples, p-benzoquinone, phenadine, methosulfate and ferrocene can be used as the electron acceptor. As the buffer, any buffer that can demonstrate a pH value which gives the highest activity to the employed enzyme such as any salts of citric acid can freely be used in addition to the phosphate buffer used in the examples. The present invention can widely be applied to any reaction system where an enzyme participates, such as alcohol sensor, sucrose sensor, and cholesterol sensor, in addition to the exemplified glucose sensor, and lactic acid sensor. In these cases, alcohol oxidase, lactic acid dehydrogenase, cholesterol oxidase, cholesterol dehydrogenase, xanthine oxidase, and an amino acid oxidase can be used in compliance with the specific substance to be quantified, in addition to the fructose dehydrogenase, lactic acid oxidase and glucose oxidase. As described in the above, the biosensor produced according to the method of the present invention can be applied to the quantification of the specific component contained in the various biological samples in a rapid and simple manner with a high accuracy. Further, since the biosensor can be stored for a long period of time, its value of utilization is great in quality control of foodstuffs as well as in clinical tests. Example 1 (Fructose Sensor I) FIG.1 is a cross-sectional side view showing a fructose sensor prepared in accordance with an embodiment of the present invention with its cover and a spacer omitted, and FIG.2 is an exploded perspective view of the fructose sensor with its reaction layer omitted. An insulating base 1 is made of polyethylene terephthalate. On the insulating base 1, there are provided lead wires 2 and 3 of silver by means of screen printing. An electrode system including a working electrode 4 and a counter electrode 5 is also formed on the insulating base 1 by printing an electrically-conductive carbon paste containing a resin binder. Further, an insulating layer 6 is formed on the insulating base 1 by printing an insulating paste. The insulating layer 6 maintains areas of the exposed regions of the working electrode 4 and the counter electrode 5 constant, and partly covers the lead wires 2 and 3. After the electrode region was prepared in this manner, a mixed aqueous solution composed of an aqueous solution (0.5 wt%) of a hydrophilic polymer, sodium salt of carboxymethyl cellulose (hereinafter referred to CMC) which dissolved fructose dehydrogenase (EC1. 1. 99. 11.; hereinafter referred to FDH) as an enzyme and potassium ferricyanide as an electron acceptor, was dropped on the electrode system. By being dried in a hot air dryer at 40°C for 10 minutes, an FDH-potassium ferricyanide-CMC layer 7 was formed. On the FDH-potassium ferricyanide-CMC layer 7, there was dropped a dispersion prepared by dispersing microcrystals of potassium dihydrogenphosphate and dipotassium hydrogenphosphate as a buffer in a toluene solution (0.5 wt%) of lecithin as a dispersing medium, which was then dried to form a buffer-lecithin layer 8. Since toluene used as the solvent for forming the layer 8 did not dissolve CMC in the underlying layer, a direct contact of the buffer in the layer 8 with the enzyme in the layer 7 was effectively avoided. Further, by the provision of the layer containing an amphipathic lipid such as lecithin on the surface of the reaction layer, an infusion of the sample solution from the surface into the reaction layer can be made with ease. As described in the above, the reaction layer of the fructose sensor was formed. The manufacturing process of the biosensor can be simplified by dropping the mixed solutions containing the hydrophilic polymer, the enzyme and the electron acceptor, each in a stroke, and by the subsequent drying. The temperature range during the drying step is preferably from 20°C to 80°C which does not lead to a deactivation of the enzyme but is sufficient for completing the drying in a short period of time. After forming the reaction layer in the above-mentioned manner, the fructose sensor was completed by adhering a cover 14 and a spacer 13 to the insulating base in a positional relationship shown by single dot-dash-lines in FIG.2. By a simple operation of bringing the sample solution to a contact with a sample supplying inlet 15 provided on a tip of the sensor, the sample solution can easily be introduced into the reaction layer region. Since the supplying amount of the sample solution is dependent on the volume of a space formed by the cover 14 and the spacer 13, there is no need of measuring the supplying amount beforehand. Further, evaporation of the sample solution can be suppressed minimum during the measurement thereby enabling a measurement of high accuracy. In FIG.2, a reference numeral 16 designates an air inlet opening provided on the cover 14. When a transparent resin is used as the material for constituting the cover 14 and the spacer 13, it is possible to easily observe the condition of the reaction layer and the state of introducing the sample solution from the outside. Two minutes after supplying 3 µl of a fructose standard solution as the sample solution to the fructose sensor thus prepared through the sample supplying inlet 15, a pulse voltage of +0.5V on the basis of the voltage at the counter electrode was applied to the working electrode. Then the anodic current value 5 seconds after the application was measured. When the sample solution reached the reaction layer, the sample solution dissolved the buffer-lecithin layer 8 to have a desirable pH value, and subsequently dissolved the FDH-potassium ferricyanide-CMC layer 7. During this process, the fructose contained in the sample solution was oxidized by the FDH, and then the potassium ferricyanide was reduced to a potassium ferrocyanide by shifting of electrons by the oxidation. Next, by the application of the above-mentioned pulse voltage, a current was generated for oxidizing the produced potassium ferrocyanide, and this current value corresponded to the concentration of fructose contained in the sample solution. The activity of the enzyme employed in the fructose sensor demonstrates its maximum value at pH4.5 at 37°C. Since the fructose standard solution is substantially neutral, when the standard solution reaches the buffer-lecithin layer 8, its pH value is adjusted to 4.5, thereby making the enzyme activity highest. Further, by separating the buffer from the enzyme, it is possible to improve the storing property of the sensor. The response obtained with the thus prepared fructose sensor to the fructose standard solution demonstrates a linear relationship for the fructose concentration, and the linear relationship can be maintained after a storing for a long period of time. In the above-mentioned example, in place of the buffer-lecithin layer 8, another buffer-hydrophilic polymer layer may be formed by spreading a solution prepared by dispersing the buffer in a solution of a hydrophilic polymer dissolved in an organic solvent which does not dissolve the CMC contained in the underlying layer, such as an ethanol solution of polyvinyl pyrrolidone, followed by drying. In the foregoing embodiments, although the electrode system was formed by means of screen printing with an electrically-conductive paint, it may alternately be formed by sputtering of platinum. In this case, the potassium ferricyanide employed as the electron acceptor can be dispensed with; in this enzyme reaction, hydrogen peroxide generated by reducing the oxygen in the substrate solution in proportion to the concentration of lactic acid (or glucose) can be detected by the platinum electrodes, thereby quantifying the concentration of the lactic acid (or glucose).

Claims: A method for producing a biosensor, comprising the steps of: forming a first layer (7) containing an enzyme and a hydrophilic polymer by using water as the medium on a surface of an insulating base (1) in close contact with an electrode system including a working electrode (4) and a counter electrode (5), which are provided on said insulating base (1); and forming a second layer (8) comprising a buffer and an amphipathic lipid, wherein said step of forming the second layer (8) comprises spreading an organic solvent solution of said amphipatic lipid on the first layer (7) by using an organic solvent that does not dissolve the hydrophilic polymer contained in the first layer and drying the spread solution, said organic solvent being dispersed with said buffer. A method for producing a biosensor in accordance with the claim 1, wherein said step of forming the first layer (7) comprises spreading an aqueous solution which dissolves the enzyme and the hydrophilic polymer on the insulating base (1) and drying the spread solution. A method for producing a biosensor in accordance with claim 1 or 2, wherein said aqueous solution which dissolves the enzyme and the hydrophilic polymer further dissolves an electron acceptor.

Inventors: FUJISAWA SATOKO, MIYAHARA MARIKO, NANKAI SHIRO, YAMAMOTO TOMOHIRO, YOSHIOKA TOSHIHIKO, FUJISAWA, SATOKO, MIYAHARA, MARIKO, NANKAI, SHIRO, YAMAMOTO, TOMOHIRO, YOSHIOKA, TOSHIHIKO
Applicants: MATSUSHITA ELECTRIC IND CO LTD, MATSUSHITA ELECTRIC INDUSTRIAL CO., LTD.
Application year: 1994
Main classification: G01N27
IPC codes: C12Q1